CpG基序联合EB病毒建立永生化B淋巴母细胞系

目的 体外建立慢性乙型肝炎患者永生化的B淋巴母细胞系(LCLs).方法 用EB病毒感染自慢性乙型肝炎患者外周血中分离的单个核细胞(PBMC),加入CpG DNA免疫调节基序以诱导B淋巴细胞增殖,环胞菌素A(CysA)抑制T淋巴细胞的活性.光学显微镜下观察LCLs的形态特征,利用流式细胞术分析LCLs膜表面分子CD19和CD23的表达水平.结果 46例患者PBMC经EBV感染4周后,42例转化成永生化B淋巴母细胞系,成功率为91.3%.转化后的B淋巴母细胞体积增大积聚成团,可进一步分裂增殖并长期传代培养.结论 CpG免疫调节基序联合EBV感染人PBMC,提高转化效率,转化后的LCL保持了成熟B淋巴细胞的生物学特性,可作为体外研究HBV特异性免疫应答的刺激细胞和靶细胞.     

EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后 ,形成永生化的B淋巴母细胞系 (B LCL)。B LCL在体外能无限增殖 ,广泛应用于医学生物学研究领域。本文就EBV对B淋巴细胞的转化机制、建立B LCL应注意的问题以及B LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述     

EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后，形成永生化的B淋巴母细胞系(B-LCL)。B-LCL在体外能无限增殖，广泛应用于医学生物学研究领域。本文就EBV对B淋巴细胞的转化机制、建立B-LCL应注意的问题以及B-LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述。     

094 EBV转化的B淋巴母细胞系的建立及应用

EBV转化B淋巴细胞后,形成永生化的B淋巴母细胞系(B-LCL).B-LCL在体外能无限增殖,广泛应用于医学生物学研究领域.本文就EBV对B淋巴细胞的转化机制、建立B-LCL应注意的问题以及B-LCL在细胞与分子遗传学、免疫学、EBV相关肿瘤的免疫治疗等方面的进展作一简要综述.     

HFRS患者汉滩病毒核蛋白特异性T细胞克隆及其靶细胞的建立

目的　建立肾综合征出血热 (HFRS)患者汉滩病毒 (HTNV)核衣壳蛋白 (NP)特异性CTL克隆 ,为HTNVNPT细胞表位鉴定及HFRS患者T细胞免疫功能研究奠定基础。方法　采用Fi coll密度梯度离心法分离HFRS患者外周血单个核细胞 (PBMC) ,用灭活HTNV和IL 2体外刺激 ,有限稀释法建立T细胞克隆 ,流式细胞术鉴定克隆表型。并用EB病毒 (EBV)转化B淋巴细胞 ,建立B淋巴母细胞样细胞系 (B LCL) ,以含HTNVS基因的重组痘苗病毒感染B LCL作靶细胞 ,以CTL克隆作效应细胞进行细胞毒杀伤试验 ,测定T细胞克隆的抗原特异性。结果　T细胞克隆能特异性识别表达NP的B LCL。在 5名患者中 ,3名有较高的杀伤率 ,并自 2名患者建立了 5株HTNV特异性CTL克隆 ,其表型为CD8 均大于 6 0 %。结论　成功建立了HFRS患者HTNVNP特异性CTL克隆及其靶细胞。NP是HFRS患者HTNV特异性CTL应答的主要靶抗原之一。     

应用环孢素(Cyclosporin A)建立EBV转化的永久人类淋巴母细胞系

人类细胞遗传学研究提示科学的进步取决于实验方法的改进,真正的临床细胞遗传学开端是以淋巴细胞培养技术为标志的,但是由于这种细胞生活周期有限,因此需反复采血进行检查。当病人由于某些原因不能继续采血时,这种细胞培养方法就不适宜了。最近,采用了一种简便的常规研究方法,即利用EB病毒(EBV)转化B淋巴细胞,并且加入环孢素(cyclosporin A,Cy A)可有效地促进EBV转化的B淋巴细胞系生长,这样就可以建成永久的淋巴母细胞系(LCL)。实验证明,利用Cy A建立淋巴母细胞系成功率达95～100%,并且同     

永生化EB病毒转化B细胞对肝癌细胞抗原的提呈作用

目的 建立EB病毒(Epstein—Barr virus，EBV)转化的永生化B细胞(命名为LEBV)并研究其对肝癌细胞抗原的提呈作用。方法 从人外周血中分离出外周血单个核细胞(Perpheral blood mononuclear cell,PBMC)，用等体积的EBV上清混合培养4周以上建立永生化EBV转化B细胞(LEBV)，用流式细胞计数仪(FACS)检测其细胞表面功能相关分子CD86、CN40、HLA-DR和HLA-ABC的表达情况。LEBV与自体淋巴细胞和肝癌细胞抗原共培养3d，结束培养前12h加入37kBq／孔^3H—标记的胸腺嘧啶(^3H—TdR)，收获细胞，用液体闪烁计数仪测定cpm。结果 培养3周以上即培养出EBV转化的永生化B细胞，可连续培养1年以上。经FACS检测，细胞表面分子CD86、CD40、HAL-DR和HLA—ABC的表达率分别为74％、91％、83％和86．7％。该细胞接触肝癌细胞抗原后刺激自体淋巴细胞增殖的能力增强。结论 EBV转化的永生化B细胞对肝癌细胞抗原具有提呈作用。     

慢性乙型肝炎PBMC凋亡及淋巴细胞亚群的检测

目的了解慢性/慢性重型乙型肝炎外周血淋巴细胞激活诱导细胞死亡(AICD)现象的存在情况、各免疫细胞的状况、AICD与淋巴细胞状况的关系,以探讨乙型肝炎慢性化和重型化的机制.方法利用慢性、慢性重型乙型肝炎病人和健康献血员外周血单个核细胞(PBMC)在PHA-P刺激下培养72h,通过流式细胞仪检测PBMC的凋亡情况;采用流式细胞仪结合全自动血液分析仪对慢性、慢性重型乙型肝炎病人及正常对照组外周血各淋巴细胞亚群进行检测.结果慢性乙型肝炎组PBMC凋亡率高于慢性重型乙型肝炎组(P＜0.01),高于正常对照组(P＜0.01).慢性乙型肝炎组总淋巴细胞百分率高于慢性重型乙型肝炎组(P＜0.01),慢性重型乙型肝炎组淋巴细胞数低于正常对照组(P＜0.01);CD3+、CD3+CD4+细胞数低于正常对照组(P＜0.01),低于慢性乙型肝炎组(P＜0.05);CD3+CD8+细胞数低于正常对照组(P＜0.05),低于慢性乙型肝炎组(P＜0.05).单核细胞百分率高于正常对照组(P＜0.01),高于慢性乙型肝炎组(P＜0.05).结论慢性乙型肝炎患者外周血淋巴细胞活化与凋亡共存,慢性重型乙型肝炎患者外周血淋巴细胞消耗严重,AICD参与乙型肝炎慢性化、重型化的发生机理.     

人类B-细胞系(BES-1)与绵羊红细胞形成玫瑰花结,产生嗜异抗体并吞噬绵羊红细胞

从人类血生成细胞建立连续培养的细胞系可通过以下两种途径:第一,可从恶性血生成细胞系建立;第二,将EB病毒(EBV)感染具有有B细胞性质的淋巴母细胞系(B LCL),可从某些非恶性B淋巴细胞建立起来。近年来通过EVV 体外感染正常B细胞,虽然能成功地建立针对已知抗原产生抗体的B-LCL,但对这些细胞产生的Ig的抗体活性却了解很少。作者建立了具有B细胞性质的B-LCL,命名为BES-1,能针对嗜异抗原(F)连续产生IgMk抗体,与SRBC能形成玫瑰花结,具有吞噬SRBC的能力。     

儿童原发性免疫缺陷病患者EBV转化的B淋巴母细胞系的建立及应用

目的: 建立原发性免疫缺陷病人外周血永生化B细胞系,保存原发性免疫缺陷病人特有的基因组资源,为进一步研究原发性免疫缺陷病提供丰富实验材料.方法: 采用密度梯度离心法分离PBMC,加入环孢菌素A、 EB病毒共培养以转化外周血淋巴细胞.结果: 成功建立14例原发性免疫缺陷病人外周血永生化B细胞系,建株成功率100%;对已建立的永生细胞株冻存和复苏,成功率为100%;转化前后制备细胞染色体和G显带核型分析无明显改变.结论: 经EB病毒成功转化外周血B淋巴细胞为永生细胞株,为进一步开展原发性免疫缺陷病的基础研究提供了资料.     

Circulating CD4+ CD25+ regulatory T cells correlate with chronic hepatitis B infection

Circulating CD4+ CD25+ regulatory T cells (Tregs) have been demonstrated to maintain immunotolerance and suppress the antigen-specific or antigen-non-specific T-cell responses, but their role in chronic hepatitis B (CHB) infection in humans has not been well characterized. In this study, we analysed the frequency and phenotypic characteristics of CD4+ CD25+ Tregs in patients of different hepatitis B virus (HBV) infection status, and investigated the effect of Tregs on antiviral immune responses in CHB patients, and the mechanism of this effect. A total of 137 subjects, including 79 CHB patients, 26 asymptomatic HBV carriers (ASCs), 12 acute hepatitis B (AHB) patients and 20 healthy controls, were enrolled in the study. We found that the frequency of CD4+ CD25(high) Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients. CD4+ CD25+ Tregs produced interleukin (IL)-10 but little or no interferon (IFN)-gamma under anti-CD3 stimulation. In CHB patients, the frequency of CD4+ CD25(high) Tregs positively correlated with serum viral load, and the Tregs were capable of suppressing the proliferation and IFN-gamma production of autologous peripheral blood mononuclear cells (PBMC) mediated by HBV antigen stimulation in vitro. However, combined administration of anti-programmed death-1 (PD-1) and anti-cytotoxic lymphocyte antigen-4 (CTLA-4) monoclonal antibody slightly enhanced the cellular proliferation and significantly increased the IFN-gamma production of PBMC cocultured with Tregs at a ratio of 2:1. Thus, the frequency of circulating CD4+ CD25+ Tregs is increased in patients with CHB, and this may play an important role in viral persistence by modulating virus-specific immune responses.     

体外诱导人类B细胞分泌HLA抗体的实验研究

目的:探索体外诱导人类B细胞产生特异性HLA抗体的理想培养条件。方法:以EBV转化致敏肾移植患者B淋巴细胞系(BLCL)为模型,采用ELISA和ELISPOT方法,比较3种培养体系:(1)RPMI1640 抗OPTI单克隆抗体(mAb);(2)RPMI1640 0·1mmol/L非必需氨基酸液 1mmol/L丙酮酸钠 40mg/L转铁蛋白;(3)Hybridoma无血清培养液,以及3种培养条件:(1)对照组(仅培养液),(2)培养液 rIL-4 rIL-10 鼠抗人CD40(α-CD40),(3)培养液 rIL-4 rIL-10 重组人CD40配体(CD40L),人类B细胞产生免疫球蛋白(Ig)和特异性抗HLA抗体的能力。结果:BLCL细胞在Hybridoma SFM培养体系所产生的IgG和IgM浓度分别为25·2～28·1mg/L和7·14～7·95mg/L,高于其他两种培养条件(P<0·05)。BLCL细胞在添加了IL-4、IL-10和α-CD40或CD40L的杂交瘤无血清培养液中能产生特异性抗HLA-I抗体,所获得的斑点频数分别为3·06/每104个BLCL细胞和3·55/每104个BLCL细胞,高于对照组(1·77/每104个BLCL细胞)。结论:BLCL细胞在杂交瘤无血清培养液中和添加了IL-4、IL-10和α-CD40或CD40L的培养条件下,提高分泌IgG和IgM的能力和特异性抗HLA-I类分子的抗体。     

HBcAg-induced upregulated 4-1BB ligand on B cells contributes to B-cell hyperactivation during chronic hepatitis B infection

During the natural history of chronic hepatitis B infection (CHB), the function of B cells is still obscure. Several limited studies have suggested that B cells are highly active in patients with CHB. In the present study, we reported that the 4-1BB ligand (4-1BBL) expression on B cells was significantly higher in patients with CHB than that in healthy subjects, meanwhile, the patients with CHB had higher serological IgG levels. Further, after being stimulated with sCD40L or hepatitis B core antigen (HBcAg), B cells had higher levels of 4-1BBL. After being cocultured with 4-1BBL+ B cells, the expressions of CD69 and 4-1BB on CD4+ T cells were significantly higher than that cocultured with 4-1BB− B cells. Cytokines including interleukin (IL)-2, IL-4, and IL-6 were significantly higher in the supernatant from 4-1BBL+ B cells coculture group than those from coculture group of 4-1BBL− B cell group, respectively; while IFN-γ and TNF-α in cocultured supernatant of 4-1BBL+ B cell group were significantly lower. Consistently, the total IgG levels in culture supernatant were significantly higher in 4-1BBL+ B cell group. Thus, hyperactive status of B cells in patients with CHB could be partially derived from the higher 4-1BBL expression on B cells triggered by HBcAg. 4-1BBL signaling pathway is involved in B cells activation, and further regulates B cell-T cell interaction by modulating the cytokines secretion, which might be critical in B cells dysfunction during CHB infection.     

Taurocholic acid inhibits the response to interferon-α therapy in patients with HBeAg-positive chronic hepatitis B by impairing CD8+ T and NK cell function

Pegylated interferon-alpha (PegIFNα) therapy has limited effectiveness in hepatitis B e-antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. However, the mechanism underlying this failure is poorly understood. We aimed to investigate the influence of bile acids (BAs), especially taurocholic acid (TCA), on the response to PegIFNα therapy in CHB patients. Here, we used mass spectrometry to determine serum BA profiles in 110 patients with chronic HBV infection and 20 healthy controls (HCs). We found that serum BAs, especially TCA, were significantly elevated in HBeAg-positive CHB patients compared with those in HCs and patients in other phases of chronic HBV infection. Moreover, serum BAs, particularly TCA, inhibited the response to PegIFNα therapy in HBeAg-positive CHB patients. Mechanistically, the expression levels of IFN-γ, TNF-α, granzyme B, and perforin were measured using flow cytometry to assess the effector functions of immune cells in patients with low or high BA levels. We found that BAs reduced the number and proportion and impaired the effector functions of CD3⁺CD8⁺ T cells and natural killer (NK) cells in HBeAg-positive CHB patients. TCA in particular reduced the frequency and impaired the effector functions of CD3⁺CD8⁺ T and NK cells in vitro and in vivo and inhibited the immunoregulatory activity of IFN-α in vitro. Thus, our results show that BAs, especially TCA, inhibit the response to PegIFNα therapy by impairing the effector functions of CD3⁺CD8⁺ T and NK cells in HBeAg-positive CHB patients. Our findings suggest that targeting TCA could be a promising approach for restoring IFN-α responsiveness during CHB treatment.     

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.     

慢性乙型肝炎患者树突状细胞体外功能恢复及诱导T细胞反应研究

目的 研究慢性乙型肝炎(CHB)患者外周血树突状细胞(DC)和T细胞体外功能,并尝试恢复功能的方法.方法 12例CHB患者作为研究对象,10例健康人作为正常对照研究.对外周血DC进行表型分析和混合淋巴细胞反应(MLR)测定,并分别用HBsAg或HBcAg作为抗原负载DC,后用ELIspot法检测产生IFN-γ的细胞频数.结果 CHB患者外周血DC共刺激分子水平表达降低,刺激MLR能力不足.经过体外补充促成熟细胞因子,DC缺陷能够得到部分纠正.成熟DC较不成熟Dc刺激MLR和诱导产生IFN-γ的T细胞能力强,HBcAS比HBsAS有更强的免疫原性.结论 慢性CHB患者外周血DC功能缺陷,能够通过补充促成熟因子纠正.成熟DC比不成熟DC显示出更强的MLR反应和诱导IFN-γ产生细胞的能力,这提示HBV蛋白负载DC作为治疗性疫苗具有可行性.     

Selection of Epstein-Barr virus specific cytotoxic T lymphocytes can be performed with B lymphoblastoid cell lines created in serum-free media

Epstein-Barr Virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) are currently used for numerous applications in cellular immunology. Where protocols destined for clinical application are concerned, the final choice of assay is made according to a risk/benefit ratio analysis. In this balance the use of xenogenic or allogenic serum has always been a major concern, as it carries both an infectious and an immunological risk. So far, it is unknown whether serum can be omitted from the entire BLCL selection procedure. In addition, as BLCL have been described as heterogeneous, serum deprivation may affect their antigen-presenting capacity. In the present study, BLCL were generated in the absence or presence of fetal calf serum (referred to as BLCL0 or BLCL(FCS), respectively). Next, in order to assess the antigen-presenting capacity of these cells, we compared the ability of BLCL0 and BLCL(FCS) cells to stimulate the EBV-specific repertoire of the corresponding donor's peripheral blood mononuclear cells in vitro. Our results showed that addition of serum was not essential for BLCL infection and culture, and that as far as we could determine, BLCL0 cells were as effective as BLCL(FCS) in reactivating the EBV-specific T-cell repertoire in vitro. Notably, FCS-specific T-lymphocytes can be detected among the BLCL(FCS)-specific CD4+-CTL. Not only was this latter observation unexpected for an EBV-seropositive donor, but it implied that the BLCL had captured and processed the corresponding FCS-derived solubles antigens; taken together our results emphasized the interest of the possibility to generate BLCL0, both for research and for clinical applications.     

Overexpression of Fc receptor-like 1 associated with B-cell activation during hepatitis B virus infection

The role of B cells in the pathogenesis of hepatitis B virus (HBV) infection has not been explored in depth. In the present study, the activation status of B cells from peripheral blood of healthy controls (N = 20) and patients with acute hepatitis B (AHB, N = 15) or chronic hepatitis B (CHB, N = 30) was evaluated by measuring the expression levels of B-cell activation markers CD69 and CD86, using quantitative real-time PCR and flow cytometry. Moreover, the potential mechanism underlying B-cell activation during HBV infection was further investigated by analyzing the expression profile of FCRL1, an intrinsic activation molecule of B cells. An elevation in the levels of B-cell activation markers including CD69 and CD86 was observed in the AHB patients (44.31 ± 9.27, 27.64 ± 9.26%) compared to CHB patients (30.35 ± 11.27, 18.41 ± 6.56%, P < 0.05), which was still higher than healthy controls (12.23 ± 7.84, 8.22 ± 3.43%, P < 0.05). Furthermore, the expression of FCRL1 was found to be similar to B-cell activation markers, which was highest in AHB patients (70.15 ± 17.11%), lowest in healthy donors (36.32 ± 9.98%, P < 0.05) and half-way between these levels in patients with CHB (55.17 ± 12.03%, P < 0.05). The results were positively associated with aberrant B-cell activation. These data suggest that B cells can play a role in HBV infection, and therefore more effort should be devoted to exploring their functions.     

重型乙型肝炎前期患者外周血免疫活性细胞的特点

目的 分析重型乙型肝炎前期(以下简称重肝前期)患者外周血免疫活性细胞的特点.方法 选取重肝前期患者48例,慢性乙型肝炎患者35例,健康志愿者20例,采用流式细胞仪检测外周血淋巴细胞CD3+、CD3+/CD4+、CD3 +/CD8+、CD4 +/CD25 +/CD45+等亚群表达百分比,计算各淋巴细胞亚群绝对值,并进行统计学分析.结果 与慢性乙肝组及健康对照组相比,重肝前期组CD3+T细胞、CD8+T细胞、CD4+ CD25+调节性T细胞(Tregs)的绝对值均明显下降(P＜0.01或P＜0.05);重肝前期组CD4+T细胞绝对值与慢性乙肝组差异无统计学意义(P＞0.05),但CD4+T细胞百分率明显高于慢性乙肝组(P＜0.05).此外,CD4 +/CD8+比值各组间存在显著差异,重肝前期组显著高于慢性乙肝组和健康对照组(P＜0.01或P＜0.05).结论 重型乙型肝炎前期存在一定程度的细胞免疫功能紊乱,其特征为CD4+T细胞占优势,CD8+T细胞和CD4+ CD25+ Tregs绝对值下降.